US 6613565 B1
Primitive hematopoietic stem cells are closely associated with discrete in vivo microenvironments. These “niches” are thought to provide the molecular signals that mediate stem cell differentiation and self renewal. We have dissected the fetal liver microenvironment into distinct cellular components by establishing an extensive panel of stromal cell lines. One particular cell line maintains repopulating stem cells for prolonged in vitro culture periods. A subtraction cloning strategy has yielded a cDNA which encodes a cell surface glycoprotein with a restricted pattern of expression among stromal cell lines. This molecule, previously identified as dlk/Pref-1, contains EGF-like repeats which are related to those in the Notch/Delta/Serrate family of proteins. We have investigated the potential role of this molecule in hematopoietic stem/progenitor cell regulation. We show that the dlk protein displays activity on purified stem cells by promoting the formation of “cobblestone areas” of proliferation. These cobblestone areas contain both primitive high-proliferative potential progenitors as well as in vivo repopulating stem cells.
1. A method for inhibiting the differentiation of hematopoietic stem cells, comprising contacting the stem cells in vitro or ex vivo with an amount of dlk protein sufficient to inhibit the differentiation of the stem cells, wherein the dlk protein is at least about 95% identical to the sequence shown as SEQ ID NO:10 or SEQ ID NO: 12.
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15. A method for enriching a population of stem cells in a mixture of stem cells and non-stem cells comprising contacting the mixture in vitro or ex vivo with an amount of human dlk protein sufficient to inhibit the differentiation of the stem cells, wherein the stem cells are hematopoietic stem cells and the dlk protein is at least about 95% identical to the sequence shown as SEQ ID NO: 12.
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This application is a CIP of Ser. No. 08/612,719 filed Mar. 8, 1996 abandoned, which is a CIP of Ser. No. 08/609,533, filed Mar. 1, 1996 abandoned.
The present invention is directed to the use of Delta-like protein (dlk) to inhibit the rate of differentiation of stem cells.
The differentiated cells of some biological systems mature in stages from a common progenitor cell, usually called a stem cell. Such cells include, for example, hematopoietic, neural, epithelial, endothelial, and mesodermal cells.
Stem cells are able to differentiate into mature cells within one of these systems. Differentiation may occur through uncommitted or committed progenitor cell intermediates.
The differentiation of a stem cell may result in one mature cell. Alternatively, a stem cell may differentiate into multiple mature lineages within a biological system, in which case the stem cell is said to be pluripotent. In some cases, the differentiation of a stem cell may result in all the mature lineages of an entire biological system, in which case the stem cell is said to be totipotent. Stem cells are also be able to self-renew. The self-renewal must be delicately balanced by differentiation in order to maintain a healthy level of stem cells.
For example, hematopoietic stem cells constitute approximately 0.01% of the cells in adult bone marrow. These stem cells, which can be recognized by the presence of the CD34 antigen, are found in micro-environments associated with stromal cells.
Hematopoietic stem cells are induced by various cytokines, such as c-kit and flk-2/flt-3 ligand, to differentiate into increasingly lineage-committed progenitors. These progenitors differentiate further into the various mature white blood cells, red blood cells, and platelets of the hematopoietic system.
Self-renewal of the stem cells requires their maintenance in an undifferentiated state. There are various applications for methods that increase the time during which stem cell populations remain undifferentiated in vivo and ex vivo. Such applications include, for example, gene therapy, and concentrating stem cell populations.
Another application for maintaining stem cells in an undifferentiated state is cell and tissue transplantation between animals. An example of this application, in the case of hematopoietic cells, is bone marrow transplantation.
Attempts to maintain and expand stem cells, especially hematopoietic stem cells, using various cytokines and cytokine mixtures have been unsuccessful. An object of the present invention is to provide methods for the maintenance of stem cells in an undifferentiated state. A more specific object is to provide methods for the maintenance of hematopoietic stem cells in an undifferentiated state. Another object is to provide a method for enriching a population of stem cells in a mixture of stem cells and non-stem cells.
These, and other, objects as will be apparent to those having ordinary skill in the art have been met by providing a method for inhibiting the differentiation of stem cells, such as hematopoietic stem cells; and a method for enriching a population of stem cells in a mixture of stem cells and non-stem cells. The methods comprise contacting the stem cells, or the mixture of stem cells and non-stem cells, with a sufficient amount of human delta-like protein (dlk).
Preferred embodiments of the invention have been chosen for purposes of illustration and description, but are not intended in any way to restrict the scope of the invention. Preferred embodiments of certain aspects of the invention are shown in the accompanying drawings, wherein:
FIG. 1 shows the mouse dlk amino acid sequence (SEQ ID NO: 10) and the human dlk amino acid sequence (SEQ ID NO: 12);
FIG. 2 shows the mouse dlk nucleotide sequence (SEQ ID NO: 11); and
FIG. 3 shows the human dlk nucleotide sequence (SEQ ID NO: 13).
The present invention relates to a method for inhibiting the differentiation of stem cells. For the purpose of this application, inhibiting the differentiation of stem cells means preventing, or inhibiting the rate of, the differentiation of stem cells.
A stem cell refers to any cell that is capable of self renewal, and of differentiating into committed progenitors of one or more lineages within a group of cells of a biological system. Such cells include, for example, hematopoietic, neural, epithelial, endothelial, and mesodermal cells. Preferably, the stem cells are capable of re-populating at least one lineage, preferably multiple lineages, and more preferably all lineages of a biological system of cells in an ablated mammal. The method involves contacting the hematopoietic stem cells in vivo or ex vivo with a sufficient amount of delta-like protein (dlk).
The dlk protein and gene is described by Laborda et al. in the Journal of Biological Chemistry 268, 3817-3820 (1993); Lee et al. in Biochimica et Biophysica Acta 1261, 223-232 (1995); and Laborda, International PCT Application WO 94/13701. The dlk gene and protein can be isolated and purified as described in the Laborda et al. and Lee et al. articles and in the Laborda patent application.
The dlk gene and protein may originate from any mammalian source. The human gene and protein are preferred. The mouse and human dlk amino acid sequences are shown in FIG. 1. The mouse and human dlk nucleotide sequences are shown in FIGS. 2 and 3, respectively.
In addition, the mouse and human dlk amino acid sequences are given in FIGS. 1A and 1B, respectively, of the Laborda patent application; the sequence of mouse and human dlk nucleotide sequence is given in FIGS. 3 and 2, respectively, of the Laborda patent application; and the amino acid and nucleotide sequences of human dlk are given in FIG. 6 of the Lee et al. article.
The dlk gene and protein useful in the present invention may contain mutations and polymorphisms in the sequences as described above. Preferably, the dlk gene and protein are at least about 75% identical, preferably at least about 85%, more preferably at least about 95%, and most preferably at least about 99% identical to the native nucleotide or amino acid sequence of a mammal, such as of the mouse and human sequences described above.
As shown in FIG. 2 of the Laborda et al. article, dlk is a transmembrane protein that contains six EGF-like repeats in an N-terminal extracellular domain and a short C-terminal intracellular domain separated by the transmembrane domain.
The dlk protein useful in the present invention may be a full length protein. Alternatively, the dlk may be in a soluble form (sdlk). The soluble protein may comprise the entire extracellular domain, or at least enough of the extracellular domain for the molecule to retain its biological activity. The extracellular domain should comprise at least three EGF repeats (for example, EGF repeats 1-3, 2-5, or 3-6); preferably at least four EGF repeats (for example, EGF repeats 1-4, 2-5, or 3-6); more preferably five EGF repeats (for example, EGF repeats 1-5, or 2-6); and most preferably all six EGF repeats. The EGF repeats are shown in FIG. 2 of the Laborda et al. article.
The soluble dlk protein preferably lacks both the transmembrane domain and the intracellular domain. Alternatively, the soluble dlk protein may contain all or part of the transmembrane domain and either no part of the intracellular domain, or as much of the intracellular domain that still permits the dlk protein to retain its solubility in water at room temperature.
Soluble forms of dlk can be produced by methods known in the art. For example, the soluble dlk protein may be produced by cloning an appropriate DNA sequence in a suitable expression vector. The DNA is transfected into a suitable host cell, such as, for example, COS, CHO, NIH 3T3, and expressed.
Optionally, the soluble dlk protein is expressed as a fusion protein. Examples of fusion protein partners to be expressed in tandem with the dlk protein include, but are not limited to, IgG protein, metallotheonine, histidine repeats, alkaline phosphatase and FLAG protein.
The dlk protein is then expressed, and preferably secreted directly into the cell media. The protein is harvested, preferably in serum-free media, from the stable expressing cell lines.
The soluble protein can be purified by methods known in the art. A suitable purification method is affinity chromotography.
The purified protein is used for any of the utilities described in the specification The protein can also be used to immunize animals for the production of anti-dlk monoclonal or polyclonal antibodies. Such antibodies can be used to identify and isolate dlk-containing cells, such as stromal cells, that express the dlk protein.
The sdlk may be used in solution. Alternatively, the sdlk may be aggregated by physical means, such as, for example, heat, pH, etc., or chemical means, such as chemical cross linking, as is well known in the art. Such “multivalent” forms of sdlk are better able to cross link the appropriate target molecules on the surface of stem cells, and to provide the appropriate biological signals. The sdlk molecules may also be coated on small, inert beads, such as latex beads for example, or on the surface of a container, such as a culture dish.
If the dlk protein used in the method of the invention contains the transmembrane region, the dlk protein may be attached to the surface of a cell, or may be independent of cells. Some cells, such as some stromal cells, contain surface dlk naturally. An example of such a cell is the stromal cell line known as AFT024.
Alternatively, the cells may not contain surface dlk naturally, but may be transfected with the dlk gene so as to express one or more surface dlk proteins. Some examples of cells suitable to be transfected with the dlk gene include COS, CHO, NIH 3T3, and BFC012.
The dlk protein and its variants, as described above, prevents, or at least inhibits the rate of, the differentiation of hematopoietic stem cells. For this purpose, the dlk protein may be attached to the surface of a stromal cell. Alternatively, the dlk protein may be attached to the surface of a non-stromal cell or may be independent of a cell, in which case it is preferably used in conjunction with stromal cells or other factors, such as those that exist on stromal cells, that, in conjunction with dlk, prevent, or at least inhibit the rate of, the differentiation of hematopoietic stem cells.
The dlk protein and its variants, as described above, may also be used along with a cocktail of growth factors. The cocktail, including the hematopoietic cocktail described below, may be replaced by, or used in conjunction with, 5-fluorouracil.
The growth factors are selected for inclusion in the cocktail so as to be specific for the type of stem cells that are being maintained in an undifferentiated state. The cocktail has the effect of enhancing the inhibition of differentiation, and of inducing the proliferation of the stem cells, leading to self-renewal.
For example, a cocktail of growth factors for hematopoietic stem cells comprises a mixture of one or more of IL-1, G-CSF, GM-CSF, c-kit ligand, IL-3, IL-6, and flk-2/flt-3 ligand. Such cocktails have been described by Moore, et al. in “Ex Vivo Expansion of CD34+ Hematopoietic Progenitors, in Gross, eds.; Advances in Bone Marrow Purging and Processing, Proceeding of the Fourth International Symposium on Bone Marrow Purging and Processing. Orlando, Fla., Wiley-Liss (1994).
The components of the cocktail, the 5-fluorouracil, and the dlk may be used at any concentration at which they are effective. The components of the cocktail and the 5-fluorouracil may, for example, be suitably used at concentrations of 0.1-100 ng/ml, preferably 1-50 ng/ml, and more preferably 5-20 ng/ml. The dlk may, for example, be suitably used at concentrations of 1 ng-100 ug/ml, preferably 0.1-100 ug/ml, more preferably 1-50 ug/ml, and most preferably 5-20 ug/ml.
The dlk protein and its variants as described above may be used on its own, or in conjunction with a cocktail of growth factors, such as a cocktail of hematopoietic growth factors, and/or 5-fluorouracil to prevent, or at least to inhibit the rate of, differentiation ex vivo. The stem cells may be found in a mixture of stem cells and non-stem cells. For example, a mixture of hematopoietic stem cells and more mature, non-stem cells occurs in peripheral blood, bone marrow or umbilical cord blood.
During the increased time the stem cells in the mixture are maintained by the dlk in an undifferentiated state, other, more mature, non-stem cells continue to differentiate, and ultimately die. In this way, the hematopoietic stem cells become enriched and concentrated in the mixture of stem cells and non-stem cells.
For example, a mixture of hematopoietic stem cells, preferably from the bone marrow, may be removed from a patient undergoing chemotherapy or radiation treatment. Patients who will benefit especially are those being treated for cancers, such as those of the white blood cells, i.e. leukemia.
For example, the patient's bone marrow is removed from the patient. The bone marrow is purged of any malignant cells, and the patient is subjected to chemotherapy or radiation treatment. The chemotherapy or radiation treatment depletes the patient's hematopoietic cells.
The hematopoietic stem cells are maintained ex vivo in an undifferentiated state with dlk, as described above. The stem cell population may be expanded at this stage.
Alternatively, the stems cells may be isolated using either negative or positive selection. An example of positive selection is the separation of a stem cell fraction from a larger cell fraction by binding the stems cells to a monoclonal or polyclonal antibody specific to a stem cell antigen, such as CD34. Such antibodies may be bound to microbeads or to a column matrix in order to facilitate the isolation of the stem cells.
An example of negative selection includes passing a cell fraction through a column in which a variety of antibodies directed against mature cell antigens are used to bind the more mature cells, allowing the stem cells lacking these antigens to be separated into a specific fraction. The antibodies may be monoclonal or polyclonal.
Following treatment, the stem cells are reinfused into the patient for self-renewal of the patient's hematopoietic system. The stem cells are preferably derived from the patient undergoing chemotherapy or radiation treatment. If necessary, the stem cells may be derived from a patient other than the patient undergoing chemotherapy or radiation treatment. The patient is a mammal, preferably a human.
Alternatively, the stem cells may be removed from a patient and subjected to gene therapy while the cells are maintained in an undifferentiated state by the dlk protein, or a variant, as described above. The presence of the dlk increases the length of time a stem cell is available for gene therapy.
In vivo, the dlk, preferably sdlk, may be used to expand the available pool of hematopoietic stem cells. Such treatment is of benefit before chemotherapy or radiotherapy for various malignancies. Increased stem cell populations would allow a more rapid re-population of all blood cells, including leukocytes and platelets, that are typically depleted during such therapies.
dlk may be administered to patients by methods known in the art. Preferably, sdlk is administered by intravenous injection, or is injected directly into the bone marrow cavity.
1. A full-length cDNA clone for human dlk (hdlk) was obtained as described above. A soluble dlk (sdlk) expression construct was prepared by PCR by truncating the cDNA before the first codon of the predicted transmembrane domain. The positive (5′) primer (Primer 1) was located starting eight nucleotides 5′ of the ATG codon, and encoded a Hind III restriction site. The negative (3′) primer (primer 2) introduced a stop codon and a Not 1 site immediately following the codon for the ultimate alanine in the extracellular domain of human dlk. The sequences of these primers are given below. The resulting PCR fragment was cloned into the eukaruotic expression vector pcDNA3 (Invitrogen Inc.). Protein expression was achieved by introducing the expression plasmid into NIH 3T3 and CHO cells and establishing permanent lines of expressing cells. In parallel, constructs directing the expression of sdlk were joined to sequences expressing the coding regions for alkaline phosphatase and for the Fc region of human 1gG1. The resulting fusion proteins (sdlk-AP and sDlk-1g) were expressed as indicated above for sdlk. These fusion proteins can be easily purified and used in several types of binding and functional assays.
The AFT024 and BFC012 stromal cell lines were characterized for their ability to support highly enriched fetal liver and adult bone marrow stem cells using both in vitro and in vivo assays. Cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 50 mM b-mercaptoethanol (BME), and maintained at 31°-33° C., 5% CO2. For long-term cocultures with hematopoietic stem cells, confluent monolayers were irradiated (20 Gy), placed in modified Dexter media (DMEM, 10% FBS, 10% horse serum, 50 mM BME, 0.1 mM hydrocortisone) and maintained at 37° C., 5% CO2 with weekly media changes. NIH 3T3 cells were obtained from ATCC.
Total RNAs from stromal cell lines were poly A+ selected, Northern blotted, and hybridized to 32P-labeled probes according to standard protocols. A 600 bp dlk cDNA clone from the AFT024 subtracted library was used as a probe. cDNA templates for RT-PCR were prepared using SuperScript reverse transcriptase according to manufacturer's protocols (GibcoBRL). Oligonucleotide primers for the dlk PCR reactions were: sense 5′- GACCCAGGCTGCCCC-3′ (SEQ ID NO: 3) and antisense 5′-GGTACTCTTGTTGAG-3′ (SEQ ID NO: 4). For analysis of dlk expression at the protein level, antisera specific for dlk was generated by immunizing rabbits with a Flag-dlk fusion protein (described below). Resultant antibodies were purified by affinity chromatography on Sepharose C1-4B to which dlk-Ig (described below) was coupled according to the manufacturer's instructions (Pharmacia Biotech Inc.). After elution and neutralization, affinity purified dlk antibodies were dialyzed against phosphate buffered saline (PBS) and protein concentration was determined by the BCA method (Pierce). Cell surface expression of dlk in native and transfected (see below) stromal cell lines was accomplished by flow cytometry with the affinity purified dlk antibody. Stromal cell lines in active growth phase were washed once with PBS/0.5 mM EDTA, and harvested by trituration in PBS/EDTA. Twice washed (PBS/3%FBS) cells were incubated with dlk antibody and a similarily prepared irrelevant control antibody. Specific labeling was developed by donkey anti-rabbit-FITC (Jackson Inmunoresearch). Stained cells were analyzed on a Becton Dickinson FACScan using Cell Quest software.
The expression plasmid pCD4-Ig contains cDNA for the extracellular domain of human CD4 fused to genomic sequences of the human immunoglobin heavy chain. This cDNA was cloned into EcoRI and Not I sites of pcDNA3 (Invitrogen) to give the plasmid KB52.3.2. cDNA encoding the extracellular domain of dlk was obtained by RT-PCR with primers BP 151 and BP 152 using total RNA from NIH 3T3 cells as template. The primer BP 152 includes the last codon in the dlk extracellular domain and contains an EcoRI site in frame with that of KB52.3.2. The resulting PCR fragment was cloned into KB52.3.2 via Hind III and EcoRI sites to obtain the soluble dlk-Ig expression plasmid. pdlk-Ig or pCD4-Ig were transfected into NIH 3T3 cells together with pSVNeo and stable clones were isolated after selection in 400 mg/ml G418 (active wt., GibcoBRL) in DMEM, 10% FBS. Primers; sense BP 151: 5′GAGGGTACCAAGCTTCGTGGTCCGCAACCAGAAG-3′ (SEQ ID NO: 5); anti-sense BP 152: 5′CTCAGATCTGAATTCGGCCTGTCCCTCGG TGAGGAG-3′ (SEQ ID NO: 6).
The CD4-Ig and dlk-Ig proteins were harvested in serum-free media from stable expressing cell lines. The soluble fusion proteins were purified by affinity chromatography on HiTrap Protein G-sepharose (Pharmacia Biotech Inc.) equilibrated with 0.1 M Tris-HCl, pH 7.6, 0.5 M NaCl and eluted with 0.2 M glycine-HCl, pH 2.8, 0.5 M NaCl. Eluted protein was dialyzed against PBS. Protein concentration was determined by the BCA method (Pierce).
Flag-dlk fusion protein was used to immunize rabbits for the production of dlk antiserum. The protein expression plasmid pcDNA3-Flag is a modification of the plasmid pcDNA3 (Invitrogen) and contains 22 base pairs of 5′ untranslated sequence, a preprotrypsinogen signal sequence, and the coding region for the Flag peptide (DYKDDDDKI) as well as a Bgl II restriction site. A cDNA fragment encoding the extracellular domain of dlk was obtained by RT-PCR using RNA from NIH3T3 cells. The 5′ primer (BP155) was designed to introduce an in frame Bgl II site at the 5′ end of the predicted mature dlk protein coding sequence. The 3′ primer (BP154) contained Xba I and Not I sites downstream of a stop codon which is immediately adjacent to the last amino acid of the predicted dlk extracellular domain. Primers; sense BP 155: 5′-GACAAGATCTCAGCTGAATAGCGACCCACCCTGTG-3′ (SEQ ID NO: 7); antisense BP 154: 5′-GCATCTAGAGCGGCCGCTCAGGCCTGTCCCTCGGTGAGGAG-3′ (SEQ ID NO: 8). The PCR fragment was digested with Bgl II and Not I and ligated into pcDNA3-Flag to yield pFlag-dlk. pFlag-dlk was transfected into COS cells using DEAE-dextran. Affinity purification of the Flag-dlk protein from COS conditioned media was performed according to manufacturer's directions using the Flag monoclonal antibody, Ml, immobilized on agarose (International Biotechnologies).
Full-length murine dlk cDNA was obtained by RT-PCR with primers BP 151 (SEQ ID: NO 5, see above) and antisense BP 200:
using total RNA from NMH3T3 cells as template. Sequencing confirmed that the resulting product was identical to the published dlk sequence. The product was subcloned into the vector pCRII (Invitrogen) and then cloned into a retroviral expression vector via the primer-encoded HindIII and NotI sites. Supercoiled plasmid was transfected into BFC012 stromal cells by the calcium phosphate method, according to the manufacturer's protocol (GibcoBRL) together with the pZeo (Invitrogen) selectable marker and selected in 50 mg/ml Zeocin (Invitrogen). BFC012 cells were also transfected with pZeo alone and selected as above. Clones from both selected populations were isolated and all remaining colonies (100-200/dish) were pooled and expanded as populations.
Hematopoietic stem cell populations were derived from wild type, Ly5.2-C57B1/6J (Jackson Laboratories), day 14 fetal liver, enriched for the AA4.1+, Sca-1+, c-kit+, and linlo/− phenotype, by immunopanning and fluorescence-activated cell sorting. Adult bone marrow (BM) was used directly after density centrifugation and immunomagnetic bead depletion or was further enriched for Sca-1+, c-kit+, linlo/− cells by flow cytometry as described. Cell sorting and data analysis was accomplished with a Becton Dickinson FACS Vantage using Cell Quest Software. Stromal cell/stem cell cocultures were initiated in 12-well trays with 300-1000 enriched stem cells per well. Cobblestone areas were quantitated by inverted-phase microscopy as described. Clonogenic progenitor assays were performed with either freshly purified stem cells or cells harvested from the stromal cocultures. These were cultured in cytokine-containing semisolid media according to the manufacturer's recommendations (Stem Cell Technologies). Progenitor colonies were scored after 8-12 days according to established criteria. Soluble dlk and control fusion proteins were added to semisolid progenitor assays at concentrations of 0.1, 0.5 and 1.0 mg/ml and also to BFC012 stromal cocultures at concentrations of 0.1 mg/ml. Fusion protein was replenished weekly in the stromal cocultures.
Cultured cells were harvested, combined with fresh unfractionated BM obtained from congenic C57B1/6 Ly5.1 mice (National Cancer Institute) and transplanted into lethally irradiated (10 Gy, split dose 3 hours apart from a 137CS source, 1 Gy/min) Ly5.1 recipient mice. Each mouse received 2×105 competitor BM cells and a fraction of the cocultured stem cells. Mice were bled by capillary puncture of the orbital venous plexus and 100 ml were collected into heparin-containing (10 U/ml) DMEM; red blood cells were removed by NH4Cl lysis. The nucleated cells were stained for the Ly5.2 (CD45.2) allelic marker using either FITC-labeled directly conjugated Ly5.2 monoclonal antibody or a biotinylated form developed with streptavidin conjugated to Texas Red. Cells were also stained with directly conjugated antibodies to lineage markers. All antibodies and chromogens were obtained from Pharmingen. Flow cytometric analysis was done on a Becton Dickinson FACS Vantage using Cell Quest Software.
The invention as claimed is enabled in accordance with the specification and readily available references and starting materials.
Nevertheless, on Mar. 3, 1997, Applicants have deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 (ATCC) transfected cell line BFC012, under ATCC Accession Number CRL-12308. This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the regulations thereunder (Budapest Treaty). This assures maintenance of a viable culture for thirty years from date of deposit. The organisms will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Applicants and ATCC, which assures unrestricted availability upon issuance of the pertinent U.S. patent. Availability of the deposited strains is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.